Vascular endothelial cells cover the entire inner surface of blood vessels in the body. They play an important role in tissue homeostasis, fibrinolysis and coagulation, blood-tissue exchange, vascularization of normal and neoplastic tissues, and blood cell activation and migration during physiological and pathological processes. A unique aspect of endothelial cells is that although they present many common functional and morphological features, they also display remarkable heterogeneity in different organs. It has been shown that bovine aortic endothelial cells, when co-cultured with cells or matrix proteins from various organs, will change their phenotype to reflect their interaction with that particular tissue type. These phenotypes are, in part, mediated by molecular markers, which are expressed by these endothelial cells specific for the unique interaction. Based on the unique histologic appearance of tumor vasculature, it is postulated that expression of specific molecular endothelial markers probably also exist for the tumor-endothelial interaction. The ability to target these molecules would, in effect, specifically target the tumor endothelium, and hence, the tumor. Previous attempts to identify and target these specific molecular endothelial molecules have failed.
The importance of pathologic angiogenesis is well established in the clinical setting of cancer. Solid tumors are unable to grow much larger than 2 mm in diameter without a blood supply, and in order to express a malignant phenotype, tumors must induce new vessel growth. Tumors recruit endothelial cells during the process of angiogenesis. The recruited endothelial cells differentiate and express unique molecular markers specific for their association with tumor cells. The ability to identify and target these molecular markers would allow for specific targeting of the tumor vasculature as well as the tumor itself. Currently, there are no viable directed therapies targeting the endothelial cells and therapies are needed to provide novel and specific treatment alternatives.
Until recently, identification of tissue-specific endothelial markers has progressed slowly, partly because of difficulties in isolating pure populations of endothelial cells from tissues. A powerful technique utilizing phage display peptide libraries has been developed that allows for the selection of peptide sequences with desired binding specificities. In this system, peptides with as many as 109 permutations are expressed on the phage surface by fusion with a phage surface protein. The desired peptides are selected on the basis of binding to the target molecule. The strength of this technology is its ability to identify interactive proteins and other molecules without pre-existing notions about the nature of the interaction.
Using this methodology, various peptides with binding specificity to angiogenic endothelium and endothelial cells from various organs have been identified. These peptide sequences are typically three amino acids in length and the best characterized of these sequences is represented by the RGD (Arg-Gly-Asp) motif. This sequence was originally discovered in fibronectin, and later re-identified using in vivo phage peptide display library animal experiments. RGD is the cell attachment site for many other adhesive proteins.
The finding that only 3 amino acids would form an essential recognition site for cells in a very large protein was initially received with some skepticism. However, the observation was soon confirmed with regard to fibronectin and then extended to other proteins. Since then, other peptide motifs that identify and bind to specific targets on the endothelial cells of angiogenic vessels, brain, lungs, retina, and kidneys have been reported (Table 1). Also, the functional specificity of the 3-amino acid motif, NGR (Arg-Gly-Asp), has recently been described for angiogenic endothelium. It is important to point out that the reported motifs with binding to tumor endothelium are, in fact, specific for angiogenic endothelial cells and not specific for tumor-derived endothelium.
TABLE 1Reported Peptide Motifs and Targeted TissuePeptide MotifsTissue TargetRDGAngiogenicNGR  EndotheliumGSLGFELungRDVRetinaSRLBrain